This is a proposal to study one aspect of the mechanism by which retinoic acid (RA) regulates cell growth and differentiation. A metabolite of the dietary factor vitamin A, a prohormone necessary for proper juvenile development, RA is a developmental morphogen during embryogenesis and a cancer chemolherapeutic agent that can convert immature neoplastically transformed cells to a mature differentiated phenotype. The proposed studies utilize an immature, uncommitted precursor cell, HL-60 human myeloblastic leukemia cells, which undergoes GO arrest arid either myeloid or monocytic differentiation when treated with RA or vitamin D3 (D3) respectively. RA causes activation of MAPK signaling and a panoply of progressive gene expression changes culminating in the (JO arrested mature myeloid differentiated cell. RA-induced MAPK activation is unusual, diverging from prototypical peptide hormone induced mitogenic RAF/MEK/-ERK signaling. It is a consequence of a steroid-like factor. Onset of activation is slow, but once activated is persistent. There is MEK de-pendent ERK activation with RAF activation surprisingly following ERK activation. And the outcome is GO arrest and differentiation. An early response to RA is the induced expression of the BLR1/CXCRC5 serpentine receptor, which is not expressed until RA treatment. BLR 1 causes MAPK signaling, and over expression accelerates RA-induced myeloid, as well as 133-induced monocytic, differentiation, and GO arrest. We have created HL-60 sublines where BLR1 is ectopically expressed by stable transfaction, or is knocked out by homologous recombination thereby precluding RA-induced expression. The specific aims are to: 1) determine the contribution of BLR1 to RA-induced MAPK signaling, in particular the intensity and duration of the signal; 2) identify candidate transcription factors activated/deactivated by BLR1 signaling in RA-treated cells; 3) determine the downstream protein targets regulated by BLR1 among RA-regulated proteins; 4) identify features of RA-induced differentiation that are dependent on BLR1 and identify BLR1 regulated proteins seminal to them. Our previous studies have established how RA regulates BLR1 expression through a novel 5' RARE consisting of paired GT boxes. The intention is to now track the action of RA from its regulation of BLR1 to receptor signaling, induced transcription factor activation, and finally changes in gene expression. This is the prototype of a paradigm where RA induces the expression of a receptor which then signals, activates transcription factors, and causes changes in gene expression culminating in specific features of RA-induced cellular differentiation. It has the implication that the ultimate effects of RA can be governed by relevant growth factors signaling through their cognate receptors.